Expression control sequences of the P-selectin gene

Abstract

DNA molecules and methods for the regulated expression of a gene in endothelial cells or megakaryocytes, are described, wherein the 5' flanking region of the P-selectin gene, or portions thereof, is ligated to the 5' end of a gene. The DNA molecules are also used as probes for screening individuals with abnormal levels of expression of P-selectin, or for production of pharmaceutical compositions to inhibit inflammation by inhibition of expression of P-selectin. These DNA molecules can also be used to identify and isolate previously unknown proteins which are involved in regulation of gene expression.

We claim: 
     
       1. A method for the expression of a gene in cultured endothelial cells or megakaryocytes comprising fusing the 5' end of the coding sequence of the gene to the 3' end of a DNA molecule comprising a nucleotide sequence consisting of nucleotides 1 to 4863 of SEQ ID NO. 5, or portions thereof effective to promote expression of a gene in cultured endothelial cells or megakaryocytes and consisting of at least 128 nucleotides immediately 5' from the translational start site of the P-selectin gene sequence including nucleotides 4736 to 4863 of SEQ ID NO. 5, to yield a recombinant gene construct;   inserting the recombinant gene construct into cultured endothelial cells or megakaryocytes and   assaying for expression of the recombinant gene inserted into the cultured endothelial cells or megakaryocytes.   
     
     
       2. The method of claim 1 for the expression of a gene wherein the 5' end of the coding sequence of the gene is fused to the 3' end of a DNA molecule comprising a nucleotide sequence selected from the group consisting of nucleotides 1 to 4851, 3525 to 4851, 3525 to 4863, 3732 to 4866, 4163 to 4851, 4163 to 4863, 4206 to 4866, 4212 to 4866, 4405 to 4851, 4405 to 4863, 4457 to 4866, 4471 to 4866, 4505 to 4866, 4555 to 4666, 4555 to 4851, 4555 to 4863, 4615 to 4666, 4615 to 4764, 4615 to 4851, 4615 to 4861, 4615 to 4863, 4626 to 4650, 4632 to 4672, 4635 to 4646, 4641 to 4866, 4642 to 4664, 4645 to 4866, 4646 to 4657, 4650 to 4669, 4667 to 4863, 4667 to 4717, 4717 to 4863, 4727 to 4866, 4747 to 4757, 4734 to 4758, 4736 to 4764, 4761 to 4866 of SEQ ID NO. 5, and combinations thereof. 
     
     
       3. The method of claim 1 wherein the gene encodes P-selectin. 
     
     
       4. An isolated nucleic acid molecule comprising a nucleotide sequence consisting of nucleotides 1 to 4863 of SEQ ID NO. 5, or portions thereof including nucleotides 4736 to 4863 of SEQ ID NO. 5 fused to the 5' end of a gene.   
     
     
       5. The isolated nucleic acid molecule of claim 4 wherein the portions further comprise consecutive nucleotides of SEQ ID NO. 5 selected from the group consisting of nucleotides 1 to 4851, 3525 to 4851, 3525 to 4863, 3732 to 4866, 4163 to 4851, 4163 to 4863, 4206 to 4866, 4212 to 4866, 4405 to 4851, 4405 to 4863, 4457 to 4866, 4471 to 4866, 4505 to 4866, 4555 to 4666, 4555 to 4851, 4555 to 4863, 4615 to 4666, 4615 to 4764, 4615 to 4851, 4615 to 4861, 4615 to 4863, 4626 to 4650, 4632 to 4672, 4641 to 4866, 4642 to 4664, 4645 to 4866, 4646 to 4657, 4650 to 4669, 4667 to 4863, 4667 to 4717, 4717 to 4863, 4727 to 4866, 4734 to 4758, and 4761 to 4866 of SEQ ID NO. 5. 
     
     
       6. Cultured endothelial cells or megakaryocytes transfected with the DNA molecule of claim 4. 
     
     
       7. Cultured endothelial cells or megakaryocytes transfected with an expression vector comprising the DNA molecule of claim 4. 
     
     
       8. The endothelial cells or megakaryocytes of claim 6 wherein the DNA is stably incorporated into the genome of the cells. 
     
     
       9. An expression vector comprising the nucleic acid molecule of claim 4. 
     
     
       10. The expression vector of claim 9 wherein the vector is a retroviral vector.